DESCRIPTION: (Investigator's Abstract): In many human diseases, cellular proliferation occurs by the expansion of a single clone of mutated cells. The resulting monoclonality within these tissues can be studied by the use specific X-linked markers. Using these clonal markers, studies of atherosclerotic plaques in humans have suggested that such plaques arise from monoclonal expansion of smooth muscle cells; the determinant marker being electrophoretically separable isoenzymes of G-6-PD. Atherosclerotic lesions generated in the hybrid hare show impressive morphologic similarities to lesions observed in human atherosclerosis. Since the hybrid hare is heterozygous for electrophoretically separable isoenzymes of G-6-PD, it provides the opportunity to study the monoclonal origin of a disease like atherosclerosis in an animal model. Because electrophoretic analysis of tissue specimens of necessity requires destruction of tissue morphology, and since the hybrid hare plaque tissue contains large numbers of foam cells, which are monocyte-derived and thus arose from a migratory cell population, detection of smooth muscle cell monoclonality may not be possible via these techniques. This proposal focuses on the production of molecular probes which can be used to determine cellular clonality in morphologically intact tissues obtained from the hybrid hare, specifically atherosclerotic tissue. Messenger RNAs corresponding to the two hare G-6-PD isoenzymes will be separately isolated from the two parental Lepus species and used to construct cDNA libraries. The libraries will be screened with human G-6-PD cDNA probes, and the DNA inserts from positive clones will be sequenced. Regions of non-homology between the two sequences will be identified and specific oligonucleotide probes complementary to these sequences, and oligopeptides coded by these sequences, will be constructed. The oligopeptides will in turn be used to prepare antibody probes. Both sets of probes will be used to localize G-6-PD isoenzymes or their mRNA precursors in morphologically intact tissues. This will enable the determination of cellular clonality within the overall architecture of hybrid hare tissues. Specifically, the clonality of smooth muscle cells in atherosclerotic plaque tissue and underlying normal tissue will be compared.